In mammalian cell lines, 2A self-cleaving peptides are frequently used to for expression of multiple genes from a single transcript. They've got some issues, e.g., leaving some proteins as fusions, but so do all of the other options, so 2As are a pretty good choice for many situations.

I have found 2As being used in many other organisms as well, including yeast and plants, but they seem to generally not be used for bacteria. I've found a couple of articles (e.g. [1], [2]) that do claim to have successfully used 2A sequences in bacteria. If this actually worked well, however, I would expect to see it much more widely used.

Are 2A sites in fact functional in bacteria and, if so, are there any issues that inhibit their use there?


2A self cleaving peptides do apparently work in bacteria (as the papers in your question suggest), but it seems that they are not commonly used in these chassis simply because they are not needed.

For context, in prokaryotes, translation is initiated by the ribosome binding to a shine-dalgarno (SD) sequence on the mRNA. These SDs can be placed anywhere along the mRNA strand, and so multiple SDs can be present. This means that translation can begin at multiple points along the mRNA, and hence proteins can be co-expressed by one promoter by including multiple RBSs and stop codons within the expression unit (i.e. promoter-RBS-CDS1-stop-RBS-CDS2-stop-terminator).

However, in many eukaryotes, translation begins at the 5' end of the mRNA where the ribosome is assembled, and scans along the mRNA until it finds the start codon. Once the protein has been synthesised and the stop codon reached, the ribosome falls off of the mRNA. This means that if there was another CDS encoded after the first stop codon, it won't be translated.

It should be noted that it is possible for eukaryotes to generate mRNA encoding multiple proteins, but as it was assumed for a long time that this was unique to prokaryotes, there is less research for this in eukaryotes. This paper has some useful information: https://academic.oup.com/femsyr/article/2/2/215/536601.

Because of these differences, for most applications, if co-expression in bacteria is required it is much simpler to add in multiple RBSs and stop codons than to rely on something like 2A peptides which, as you mentioned, do not always work and can result in fused proteins. However in eukaryotes, techniques such as 2A peptides are important considerations for co-expression as poly-cistronic mRNA (mRNA encoding multiple proteins) seem to be much less common.

This doesn't mean that there aren't applications for 2A peptides in bacteria, but they are much more specific and hence their use doesn't turn up so much in the literature.

These wikipedia pages have some good reading and references:

Disclaimer: I don't work with eukaryotes so most of the information about gene co-expression and how common it is has been gathered from reading for this question.

  • $\begingroup$ Very interesting, thank you! This is the opposite of what I was guessing, given my past experience where eukaryotic techniques don't work in bacteria because bacteria have a more constrained toolkit. $\endgroup$
    – jakebeal
    Mar 8 at 13:50
  • $\begingroup$ A potential advantage of 2A peptides over multiple RBSs is that the former ensures a 1:1 ratio between the resulting proteins. Also note that 2A peptides are not actually cleaving the peptide chain (i.e., there is not proteolytic activity involved). "Cleavage" is instead the results of ribosome skipping. $\endgroup$
    – gaspanic
    Apr 2 at 3:40

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